The projects proposed in this application will investigate basic mechanisms of acidification of the urine, using micropuncture technology in rats. We will use the isolated perfused rat kidney to study the role of ambient pCO2 and pH on acidification rates and gradients in the proximal and distal renal tubules. One aim is to determine the basic chemical reaction supplying H+ ions for secretion. In additional experiments in the isolated perfused rat kidney, we will determine whether a specific H+ ATPase is present in the luminal border of the proximal and distal tubules by microperfusing the inhibitor DCCD into the lumen. The pH of the fluid distal to the perfusion pipet will be monitored to determine whether this enzyme, thought to be the H+ translocator is present in rat kidney. In addition, the metabolic energy source for H+ secretion will be studied by use of Vanadate, which uncouples ATP from ATPase. In intact rats, proximal renal tubular acidosis will be produced by maleic acid administration. The defect in tubular HCO3 reabsorption will be studied by micropuncture. We will examine whether the H+ ATPase has been inhibited by maleic acid or whether there is increased permeability of the epithelium to allow back-diffusion of HCO3.